Difference between revisions of "Part:BBa K3431051"

Latest revision as of 15:53, 26 October 2020

Banana_ToeholdSwitch-Regulated Invertase

Introduction

Banana_ToeholdSwitch-Regulated Invertase is a genetic device consisting of 4 basic parts: T7 promoter (BBa_I719005), banana toehold switch (BBa_K3431039), Thermotoga maritima Invertase (BBa_K3431000), and T7 terminator (BBa_K731721). The protein expression of this part is regulated by the RNA of the banana.

Components

As for the T7 promoter (BBa_I719005) and T7 terminator (BBa_K731721), they are the essential genetic elements for the PURExpress protein synthesis kit.
The mechanism of the detection is mainly based on the regulatory part, Banana Toehold Switch (BBa_K3431039). Its sequence is from a modular synthetic biology education kit, banana toehold sensor sfGFP (Biobits) ^[1] . Upon binding with banana RNA, its hairpin structure can be opened up and the ribosomes can bind with its RBS (ribosome binding site), triggering the translation process of the downstream reporter, invertase (BBa_K3431000).
The reporter invertase can convert sucrose into glucose, which can be easily measured by a personal glucose meter (PGM) Therefore, the part can be used as a detection tool.

Characterization

To further understand its functionality, 2020 iGEM CSMU-Taiwan conducted a test on it. The plasmid would be transcribed and translated with the protein synthesis kit at 37℃ for 2 hours. We would then add 5μl of 0.5M sucrose and measured the glucose concentration with the Rightest ^TM GS550 glucose meter every 10 minutes at 55℃ for an hour. In our experiment, the ON state refers to the conditions with miRNA triggers; while the OFF state means that there was no miRNA in the environment.

Figure 1. The glucose concentration in 60 minutes invertase enzymatic reaction. The green line refers to the conditions with banana triggers, while the blue line refers to the conditions without a banana trigger.

Results As shown above, glucose concentration increased with time in the ON state but remained low in the OFF state. The result suggested the regulatory function of the toehold switch.

Reference

1. Huang, A., Nguyen, P. Q., Stark, J. C., Takahashi, M. K., Donghia, N.,Ferrante, T., Dy, A. J., Hsu, K. J., Dubner, R. S., Pardee, K., Jewett, M. C., &Collins, J. J. (2018). BioBits™ Explorer: A modular synthetic biology education kit. Science advances, 4(8), eaat5105. https://doi.org/10.1126/sciadv.aat5105

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1441
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1212
Illegal BamHI site found at 1342
Illegal XhoI site found at 1413
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1013
1000
COMPATIBLE WITH RFC[1000]

@@ Line 8: / Line 8: @@
 ===Components===
 As for the T7 promoter (BBa_I719005) and T7 terminator (BBa_K731721), they are the essential genetic elements for the PURExpress protein synthesis kit.<br>
-The mechanism of the detection is mainly based on the regulatory part, Banana Toehold Switch (BBa_K3431039). Its sequence is from a modular synthetic biology education kit, banana toehold sensor sfGFP (Biobits) <sup>1</sup> . Upon binding with banana RNA, its hairpin structure can be opened up and the ribosomes can bind with its RBS (ribosome binding site), triggering the translation process of the downstream reporter, invertase (BBa_K3431000).<br>
+The mechanism of the detection is mainly based on the regulatory part, Banana Toehold Switch (BBa_K3431039). Its sequence is from a modular synthetic biology education kit, banana toehold sensor sfGFP (Biobits) <sup>[1]</sup> . Upon binding with banana RNA, its hairpin structure can be opened up and the ribosomes can bind with its RBS (ribosome binding site), triggering the translation process of the downstream reporter, invertase (BBa_K3431000).<br>
 The reporter invertase can convert sucrose into glucose, which can be easily
 measured by a personal glucose meter (PGM) Therefore, the part can be used as a detection tool.
 ===Characterization===
-To further understand its functionality, 2020 iGEM CSMU-Taiwan conducted a test on it. The plasmid would be transcribed and translated with the protein synthesis kit at 37℃ for 2 hours. We would then add 5μl of 0.5M sucrose and measured the glucose concentration with the Rightest TM GS550 glucose meter every 10 minutes at 55℃ for an hour. In our experiment, the ON state refers to the conditions with miRNA triggers; while the OFF state means that there was no miRNA in the environment.
+To further understand its functionality, 2020 iGEM CSMU-Taiwan conducted a test on it. The plasmid would be transcribed and translated with the protein synthesis kit at 37℃ for 2 hours. We would then add 5μl of 0.5M sucrose and measured the glucose concentration with the Rightest <sup><I>TM</I></sup> GS550 glucose meter every 10 minutes at 55℃ for an hour. In our experiment, the ON state refers to the conditions with miRNA triggers; while the OFF state means that there was no miRNA in the environment.
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